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双螺旋点扩散函数(double-helix point spread function, DH-PSF)显微可实现纳米尺度的三维单颗粒示踪(three-dimensional single particle tracking, 3D SPT), 被广泛应用于生命科学等领域, 但DH-PSF显微的成像景深和定位精度有限, 限制了其在活体厚样品中的应用. 为了解决此问题, 本文提出了一种基于轴向分光棱镜的多焦面DH-PSF 显微(z-splitter prism-based multifocus DH-PSF microscopy, ZPMDM)方法和系统, 通过将基于轴向分光棱镜的多焦面显微与DH-PSF相结合, 在无需扫描的情况下提高DH-PSF显微的轴向定位范围和定位精度, 解决完整活细胞内3D SPT的大景深探测难题. 通过系统标定, ZPMDM中3个通道的平均三维定位精度分别为 σ L(x, y, z)= (4.4 nm, 4.6 nm, 10.5 nm), σ M(x, y, z)= (4.3 nm, 4.2 nm, 8.2 nm), 以及 σ R(x, y, z)= (4.8 nm, 4.4 nm, 10.3 nm), DH-PSF的有效景深扩展至6 μm, 实现了大景深范围内的甘油-水混合溶液中的荧光微球示踪, 并初步研究了活巨噬细胞的吞噬现象, 进一步验证了该方法的有效性, 对于3D SPT的发展和应用具有重要意义.Double-helix point spread function (DH-PSF) microscopy can realize three-dimensional single particle tracking (3D SPT) on a nanoscale, and is widely used in life sciences and other fields. However, its imaging depth-of-field (DOF) and localization accuracy are limited, which hinders its application in thick samples in vivo. To address this issue, this paper proposes a z-splitter prism-based multifocus DH-PSF microscopy (ZPMDM) method and system to improve the DOF and localization accuracy of DH-PSF microscopy without scanning. It solves the problem of large DOF detection of 3D SPT in whole living cells. By means of systematic calibration, the average 3D localization accuracies of three channels of ZPMDM are determined to be σ L(x, y, z)= (4.4 nm, 4.6 nm, 10.5 nm), σ M(x, y, z)= (4.3 nm, 4.2 nm, 8.2 nm), and σ R(x, y, z)= (4.8 nm, 4.4 nm, 10.3 nm). And the effective DOF of the system is extended to 6 μm. Furthermore, the ZPMDM system is used to track fluorescent microspheres in a glycerol-water mixture across a large depth-of-field range. The Brownian motion of the fluorescent microspheres in the mixture solution is also investigated. The experimental results demonstrate that the errors between the experimentally obtained diffusion coefficients and the theoretically calculated diffusion coefficients are all within 10%. The reliability of the ZPMDM system in achieving single-particle 3D tracking imaging is verified in this study. The validity of the method is further verified by preliminarily investigating the phagocytosis phenomenon of live macrophages. It is of significance for the development and application of nanoscale 3D SPT. The ZPMDM system is shown in the attached figure.
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